U.S. patent number 7,122,668 [Application Number 10/487,154] was granted by the patent office on 2006-10-17 for platinum complexes and their use in therapy.
This patent grant is currently assigned to Yissum Research Development Company of the Hebrew University of Jerusalem. Invention is credited to Yechezkel Barenholz, Dan Gibson, Elena Khazanov, Yousef Najajreh.
United States Patent |
7,122,668 |
Barenholz , et al. |
October 17, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
Platinum complexes and their use in therapy
Abstract
The present invention concerns novel platinum complexes in which
at least one of the amine ligand is a non-planar heterocyclic
aliphatic amine. The platinum complexes may be in a trans or cis
configuration and were found to posses therapeutic activites. Thus,
the present concerns novel platinum complexes, pharmaceutical
compositions comprising them and other uses thereof.
Inventors: |
Barenholz; Yechezkel
(Jerusalem, IL), Gibson; Dan (Jerusalem,
IL), Najajreh; Yousef (Beit Jala, IL),
Khazanov; Elena (Beit Shemesh, IL) |
Assignee: |
Yissum Research Development Company
of the Hebrew University of Jerusalem (Jerusalem,
IL)
|
Family
ID: |
23218625 |
Appl.
No.: |
10/487,154 |
Filed: |
August 21, 2002 |
PCT
Filed: |
August 21, 2002 |
PCT No.: |
PCT/IL02/00687 |
371(c)(1),(2),(4) Date: |
November 17, 2004 |
PCT
Pub. No.: |
WO03/017998 |
PCT
Pub. Date: |
March 06, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050090478 A1 |
Apr 28, 2005 |
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Current U.S.
Class: |
544/225; 548/101;
546/2; 556/137 |
Current CPC
Class: |
A61P
43/00 (20180101); A61P 35/00 (20180101); C07F
15/0093 (20130101) |
Current International
Class: |
C07F
15/00 (20060101); A61K 31/28 (20060101) |
Field of
Search: |
;556/137 ;514/492
;548/101 ;546/2 ;544/225 |
References Cited
[Referenced By]
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0 273 315 |
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Jul 1988 |
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EP |
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0727 430 |
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Aug 1996 |
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EP |
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|
Primary Examiner: Nazario-Gonzalez; Porfirio
Attorney, Agent or Firm: nath & Associates PLLC Nath;
Gary M. Hopkins; Susanne M.
Claims
The invention claimed is:
1. A platinum complex in trans configuration, the complex having
the general formula (I): [Pt(X)(Y)(Am.sub.1)(Am.sub.2)] (I)
wherein: X and Y, which may be the same or different, represent a
halogen, carboxylate, phosphate or sulphate group; Am.sub.1
represents an amine selected from ammonia, a primary amine, a
secondary amine, a non-planar heterocyclic aliphatic amine or a
heterocyclic aromatic amine; and Am.sub.2 represents a non-planar
heterocyclic aliphatic amine; provided that
trans-[Pt(piperidine).sub.2Cl.sub.2] and
trans-[Pt(morpholine).sub.2Cl.sub.2] are excluded.
2. The complex of claim 1, in the form of a dimer in which each
monomeric unit is a Pt-complex as defined in claim 1, bound to the
other Pt-complex, independently, through the Am.sub.1 or through
the Am.sub.2 or through a linker connected to said Am.sub.1 or
Am.sub.2.
3. The complex of claim 1, wherein said X and Y are the same or
different and represent chloride or iodide.
4. The complex of claim 1, wherein said X and Y both represent a
chloride.
5. The complex of claim 1, wherein said Am.sub.1 represents
ammonia.
6. The complex of claim 1, wherein said Am.sub.1 represents a
primary amine selected from methylamine, ethylamine, n-propylamine,
isopropylamine, n-butylamine, n-hexylamine, n-heptylamine or
n-nonylamine.
7. The complex of claim 1, wherein said Am.sub.1 represents a
secondary amine selected from dimethylamine, diethylamine,
dipropylamine, dibutylamine.
8. The complex of claim 1, wherein said Am.sub.1 represents a
non-planar heterocyclic aliphatic amine selected from, piperazine,
2-methylpiperazine, piperidine, 2-, 3-, or 4-hydroxypiperidine,
4-piperidino-piperidine, pyrrolidine, 4-(2-hydroxyethyl)piperazine
and 3-aminopyrolidine.
9. The complex of claim 1, wherein said Am.sub.1 represents a
heterocyclic aromatic amine selected from pyridine, 2-, 3-,
4-aminopyridine, 2-, 3-, or 4-picoline, quinoline, 3-, or
4-aminoquinoline, thiazole, imidazole, 3-pyrroline, pyrazine,
2-methylpyrazine, 4-aminoquinaldine.
10. The complex of claim 1, wherein said Am.sub.2 represents a
non-planar heterocyclic amine selected from piperazine,
2-methylpiperazine, piperidine, 2-, 3-, or 4-hydroxypiperidine,
4-piperidino-piperidine, pyrrolidine, 4-(2-hydroxyethyl)piperazine
and 3-aminopyrolidine.
11. The complex of claim 1, selected from
trans-[PtCl.sub.2(NH.sub.3)(piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4-hydroxypiperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4-piperidino-piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4,4'-bipiperidine)];
trans-[PtCl.sub.2(4-picoline)(piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl;
trans-[ptcl.sub.2(isopropylamine)(piperazine)].HCl;
trans-[PtCl.sub.2(n-butylamine)(piperazine)].HCl;
trans-[PtCl.sub.2(n-nonylamine)(piperazine)].HCl
trans-[PtCl.sub.2(piperidine)(piperazine)].HCl;
trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl;
trans-[PtCl.sub.2(piperazine)(piperazine)].HCl;
trans-[PtCl.sub.2(NH.sub.3)[4-(2-hydroxyethyl)piperazine)].HCl.
12. The complex of claim 1, being positively charged.
13. The complex of claim 2, wherein said linker comprises
4,7,10-trioxa-1,13-tridecane chain.
14. The complex of claim 12, being Bis-[{trans,
trans-(PtCl.sub.2piperazine).sub.2}(4,7,10-trioxa-1,13-tridecanediamine)]-
.2HCl.
15. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and as an active ingredient a therapeutically
effective amount of a platinum (Pt) complex in the trans
configuration, the complex having the general formula:
[Pt(X)(Y)(Am.sub.1)(Am.sub.2)] (I) wherein X and Y, which may be
the same or different, represent a halogen, carboxylate, phosphate
or sulphate group; Am.sub.1 represents an amine selected from
ammonia, a primary amine, a secondary amine, a non-planar
heterocyclic aliphatic amine or a heterocyclic aromatic amine; and
Am.sub.2 represents a non-planar heterocyclic aliphatic amine,
provided that trans-[Pt(piperidine).sub.2Cl.sub.2] and
trans-[Pt(morpholine).sub.2Cl.sub.2] are excluded.
16. The composition of claim 15, wherein the active ingredient is
said Pt complex in a form of a dimer in which each monomeric unit
is a Pt-complex bound to the other Pt-complex, independently,
trough the Am.sub.1 or through the Am.sub.2 or trough a linker
connected to said Am.sub.1 or Am.sub.2.
17. The composition of claim 15, wherein the active ingredient is
said Pt complex in which X and Y are the same or different and
represent chloride or iodide.
18. The composition of claim 17, wherein the active ingredient is
said Pt complex in which X and Y both represent a chloride.
19. The composition of claim 15, wherein the active ingredient
comprises said Pt complex in which said Am.sub.1 represents
ammonia.
20. The composition of claim 15, wherein the active ingredient
comprises said Pt complex in which Am.sub.1 represents a primary
amine selected from methylamine, ethylamine, n-propylamine,
isopropylamine n-butylamine, n-hexylamine, n-heptylamine or
n-nonylamine.
21. The composition of claim 15, wherein the active ingredient
comprises said Pt complex in which Am.sub.1 represents a secondary
amine selected from dimethylamine, dimethylamine, dipropylamine,
dibutylamine.
22. The composition of claim 15, wherein the active ingredient
comprises said Pt complex in which Am.sub.1 represents a
heterocyclic aromatic amine selected from pyridine, 2-, 3-, or
4-aminopyridine, 2-, 3-, or 4-picoline, quinoline, 3-, or
4-aminoquinoline, thiazole, imidazole, 3-pyrroline, pyrazine,
2-methylpyrazine 4-aminoquinaldine.
23. The composition of claim 15, wherein the active ingredient
comprises said Pt complex in which Am.sub.1 represents a non-planar
heterocyclic aliphatic amine selected from piperazine,
2-methylpiperazine, 2-pyrazoline, piperidine, 2-, 3-, or
4-hydroxypiperidine, 4-piperidino-piperidine, pyrrolidine,
4-(2-hydroxyethyl)piperazine or 3-aminopyrolidine.
24. The composition of claim 15, wherein the active ingredient
comprises said Pt complex in which Am.sub.2 is a non-planar
heterocyclic aliphatic amine selected from piperazine,
2-methylpiperazine, 2-pyrazoline, piperidine, 2-, 3-, or
4-hydroxypiperidine, 4-piperidino-piperidine, pyrrolidine,
4-(2-hydroxyethyl)piperazine or 3-aminopyrolidine.
25. The composition of claim 15, wherein said active ingredient is
selected from trans-PtCl.sub.2(NH.sub.3)(piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4-hydroxypiperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4-piperidino-piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4,4'-bipiperidine)];
trans-[PtCl.sub.2(4-picoline)(piperidine)];
trans-PtCl.sub.2(NH.sub.3)(piperazine)].HCl;
trans-[ptcl.sub.2(isopropylamine)(piperazine)].HCl;
trans-[PtCl.sub.2(n-butylamine)(piperazine)].HCl;
trans-[PtCl.sub.2(n-nonylamine)(piperazine)].HCl
trans-[PtCl.sub.2(piperidine)(piperazine)].HCl;
trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl;
trans-[PtCl.sub.2(piperazine)(piperazine)].HCl;
trans-[PtCl.sub.2(NH.sub.3)[4-(2-hydroxyethyl)piperazine)].HCl.
26. The composition of claim 16, wherein said active ingredient is
said dimmer in which the linker comprises a
4,7,10-trioxa-1,13-tridecane chain.
27. The composition of claim 26, wherein said active ingredient is
Bis-[{trans, trans-(PtCl.sub.2piperazine).sub.2}(4,7,10-trioxa-
1,13-tridecanediamine)]2HCl.
28. The composition of claim 15, wherein said active ingredient is
loaded onto liposomes.
29. The composition on of claim 25, wherein said active ingredient
is loaded onto liposomes.
30. A method for achieving a therapeutic effect, the method
comprising administering to a subject in need an amount of a
Pt-complex in trans configuration, the amount being sufficient for
achieving said therapeutic effect and the Pt complex has the
general formula (I): [Pt(X)(Y)(Am.sub.1)(Am.sub.2)] (I) wherein: X
and Y, which may be the same or different, represent a halogen,
carboxylate, phosphate or sulphate group; Am.sub.1 represents an
amine selected from ammonia, a primary amine, a secondary amine, a
non-planar heterocyclic aliphatic amine or a heterocyclic aromatic
amine; and Am.sub.2 represents a non-planar heterocyclic aliphatic
amine, provided that trans- [Pt(piperidine).sub.2Cl.sub.2] and
trans- [Pt(morpholine).sub.2Cl.sub.2] are excluded.
31. The method of claim 30, wherein said Pt-complex is in a form of
a dimer in which each monomeric unit is a Pt-complex bound to the
other complex, independently, through the Am.sub.1, through the
Am.sub.2 or through a linker connected to said Am.sub.1 or
Am.sub.2.
32. The method of claim 30, wherein said subject is administered
with a Pt complex in which X and Y are the same or different and
represent chloride or iodide.
33. The method of claim 30, wherein X and Y both represent a
chloride.
34. The method of claim 30, wherein said subject is administered
with a Pt complex in which Am.sub.1 represents ammonia.
35. The method of claim 30, wherein said subject is administered
with a Pt complex in which Am.sub.1 represents a primary amine
selected from methylamine, ethylamine, n-propylamine,
isopropylamine, n-butylamine, n-hexylamine, n-heptylamine or
n-nonylamine.
36. The method of claim 30, wherein said subject is administered
with a Pt complex in which Am.sub.1 represents a secondary amine
selected from dimethylamine, diethylamine, dipropylamine,
dibutylamine.
37. The method of claim 30, wherein said subject is administered
with a Pt complex in which Am.sub.1 represents a heterocyclic
aromatic amine selected from pyridine, 2-, 3- or 4-picoline,
quinoline, 3- or 4-aminoquinoline, thiazole, 2-, 3- or
4-aminopyridine, imidazole, 3-pyrroline, pyrazine, 2-methylpyrazine
or 4-aminoquinaldine.
38. The method of claim 30, wherein said subject is administered
with a Pt complex in which Am.sub.1 represents a non-planar
heterocyclic amine selected from piperazine, 2-methylpiperazine,
piperidine, 2-, 3-, or 4-hydroxypiperidine,
4-piperidino-piperidine, pyrrolidine, 4-(2-hydroxyethyl)piperazine
or 3-aminopyrolidine.
39. The method of claim 30, wherein Am.sub.2 represents a
non-planar heterocyclic amine selected from piperazine,
2-methylpiperazine, piperidine, 2-, 3-, or 4-hydroxypiperidine,
4-piperidino-piperidine, pyrrolidine,
4-(.sub.2-hydroxyethyl)piperazine or 3-aminopyrolidine.
40. The method of claim 30, comprising administering to said
subject a platinum complex selected from:
trans-[PtCl.sub.2(NH.sub.3)(piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4-hydroxypiperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4-piperidino-piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(1,4'-bipiperidine)];
trans-[PtCl.sub.2(4-picoline)(piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl;
trans-[ptcl.sub.2(isopropylamine)(piperazine)].HCl;
trans-[PtCl.sub.2(n-butylamine)(piperazine)].HCl;
trans-[PtCl.sub.2(n-nonylamine)(piperazine)].HCl
trans-[PtCl.sub.2(piperidine)(piperazine)].HCl;
trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl;
trans-[PtCl.sub.2(piperazine)(piperazine)].HCl;
trans-[PtCl.sub.2(NH.sub.3)
[4-(2-hydroxyethyl)piperazine)].HCl.
41. The method of claim 31, wherein said linker comprises a
4,7,10-trioxa- 1,13-tridecane chain.
42. The method of claim 41, wherein the subject is administered
with a therapeutically effective amount of Bis-[{trans,
trans-(PtCl.sub.2piperazine).sub.2}(4,7,10-trioxa-1,13-tridecanediamine)]-
.2HCl.
43. The method of claim 30, for achieving a therapeutic effect, the
therapeutic effect comprises forming an adduct between said Pt
complex and DNA.
44. The method of claim 31, for achieving a therapeutic effect, the
therapeutic effect comprises, forming an adduct between said Pt
complex and DNA.
45. The method of claim 30, for achieving a therapeutic effect, the
therapeutic effect comprises inhibiting undesired cell
proliferation.
46. The method of claim 31, for achieving a therapeutic effect, the
therapeutic effect comprises inhibiting undesired cell
proliferation.
47. The method of claim 45 for inducing apoptosis of undesired
cells.
48. The method of claim 46 for inducing apoptosis of undesired
cells.
49. The method of claim 30, wherein said Pt complex is loaded onto
a liposome.
50. The method of claim 31, wherein said Pt complex is loaded into
a liposome.
51. A platinum complex of the general formula (I):
[Pt(X)(Y)(Am.sub.1)(Am.sub.2)] (I) wherein: X and Y, which may be
the same or different, represent a halogen, carboxylate, phosphate
or sulphate group; Am.sub.1 represents an amine selected from
ammonia, a primary amine, a secondary amine, a non-planar
heterocyclic aliphatic amine or a heterocyclic aromatic amine; and
Am.sub.2 represents a non-planar heterocyclic aliphatic amine;
provided that the following compounds are excluded:
cis-[PtCl.sub.2(quinoline)(piperidine)];
cis-[PtCl.sub.2(piperidine)(pyridine)];
cis-[PtCl.sub.2-(piperidine)(o-CH.sub.3--C.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(piperidine)(p-CH.sub.3--C.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(morpholine)(pyridine)];
cis-[PtCl.sub.2(morpholine)(o-CH.sub.3--C.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(morpholine)(p-CH.sub.3--C.sub.6H.sub.4--NH.sub.2];
cis-[PtCl.sub.2(piperidine)(aniline)];
cis-[PtCl.sub.2(piperidine)(o-CH.sub.3O--C.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(piperidine)(p-C.sub.2H.sub.5OC.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(quinoline)(cyclohexylamine)];
cis-[PtCl.sub.2(quinoline)(morpholine)];
cis-[PtCl.sub.2(quinoline)(piperidine)];
cis-[PtBr.sub.2(piperazine)(piperazine),
cis-[PtCl.sub.2(piperazine)(piperazine)];
cis-[PtCl.sub.2(piperidine)(piperidine)];
cis-[PtCl.sub.2(morpholie)(morpholine)];
cis-[PtCl.sub.2(pyrrolidine)(NH.sub.3)],
cis-[PtI.sub.2(pyrrolidine)(NH.sub.3)],
cis-{PtICl.sub.2(pyrrolidine)(NH.sub.3)],
cis-[PtCl.sub.2(piperidine)(NH.sub.3)],
cis-[PtI.sub.2(piperidine)(NH.sub.3)],
cis-[PtCl.sub.2(piperidone)(NH.sub.3)],
cis-[PtI.sub.2(piperidone)(NH.sub.3)],
cis-[PtICl(piperidone)(NH.sub.3)],
cis-[PtCl.sub.2(3-hydroxypyrrolidine(NH.sub.3)],
cis-[PtI.sub.2(3-hydroxypyrrolidine)(NH.sub.3)],
cis-[PtClI(3-hydroxypyrrolidine)(NH.sub.3),
trans-[Pt(piperidine).sub.2Cl.sub.2]; and
trans-[Pt(morpholine).sub.2Cl.sub.2].
Description
FIELD OF THE INVENTION
The present invention relates to novel platinum complexes and their
uses.
BACKGROUND OF THE INVENTION AND PRIOR ART
Cisplatin, cis-[PtCl.sub.2(NH.sub.3).sub.2] is one of the three
most widely used clinical agents in the treatment of a variety of
solid tumors.sup.(1). It is believed to kill tumor cells by binding
irreversibly to the DNA, mainly to two adjacent guanines on the
same strand, inducing a kink in the DNA that is recognized by
cellular proteins that bind the cisplatin-modified DNA.sup.(2). It
is the Pt-DNA adducts that are responsible for the induction of
apoptosis and eventual cell death.sup.(3). Despite its efficacy in
the treatment of various neoplastic diseases, including testicular
and ovarian tumors, it's clinical utility is restricted by its low
solubility, toxicity and especially tumor resistance.sup.(4).
Second generation drugs such as carboplatin
(Pt(CBDCA)(NH.sub.3).sub.2, CBDCA=1,1-cyclobutanedicarboxylate)
exhibit reduced nephrotoxicity but fail to overcome the tumor
resistance probably due to the fact that they form the same
spectrum of DNA adducts as does cisplatin.sup.(5).
Overcoming the resistance is one of the major goals in the
development of novel platinum drugs and hence new compounds that
deviate from the classic structure-activity relationship (SAR) have
been designed, synthesized and screened.sup.(6). The SAR, first
formulated by Cleare and Hoeschele, influenced medicinal chemists
to direct their efforts to the preparation of neutral platinum(II)
complexes with two inert ligands in the cis configuration and two
semi-labile leaving groups.sup.(7).
It was generally accepted that a cis configuration of the two
leaving groups is essential for anti-tumor activity of
cis-diaminedichloroplatinum (cis-DDP). This was the situation for
more than two decades until Farrell et. al have reported that
replacing one or both NH.sub.3 ligands in
trans-PtCl.sub.2(Am.sub.1)(Am.sub.2), wherein Am.sub.1,
Am.sub.2.dbd.NH.sub.3, or planar amine ligands such as quinoline,
thiazole, pyridine or benzothiazole, (e.g.
trans-[PtCl.sub.2(NH.sub.3)(pyridine)],
trans-[PtCl.sub.2(NH.sub.3)(thiazole)],
trans-[PtCl.sub.2(NH.sub.3)(quinoline)], and
trans-[PtCl.sub.2(NH.sub.3)(benzothiazole)]) substantially enhances
the cytotoxicity of the trans geometry.sup.(8). Nguyen, H. D. et
al. Vietnam J. of Chem. (2001) 39(4), 111 114 describe the
synthesis of cis-PtCl.sub.2 complexes containing quinoline and
primary and secondary amines and discuss their .sup.1H-NMR spectra.
Tran, T. D. et al. Tap Chi Duoc Hoc (2001) 6, 6 8 describe
cis-PtCl.sub.2 complexes containing two amine-containing ligands,
one being an aromatic pyridine or benzylamines and the other a
aliphatic cyclic amine (morpholine or piperidine) and discuss the
complexes IR and Raman spectra. Tran T. D. et al. Vietnam J. of
Chem. (2001) 39(3), 99 102 describe the synthesis of cis-PtCl.sub.2
complexes containing piperidine and aromatic amine or an amine
substituted with an aromatic amine, their IR, Raman and UV spectra.
Furthermore, the complexes were tested for cell cytotoxicity on
human liver cancer cells. Tran, T. D. et al. Tap Chi Duoc Hoc
(Vietnam J. of Chem.) (1997) 35(2), 21 23 describe the synthesis of
cis-PtCl.sub.2 complexes containing guinoline and an amine selected
from morpholine, cyclohexylamine, piperidine or benzylamine, their
UV and IR spectra and their biological activity for reducing the
germination of kernels. Jonson Matthey Pub. Ltd. Co. EP-A-0727430
(1996) describes cis-Pt complexes of formula Pt(X)(Z)(A).sub.2 and
their activity against cancer cells. A is a leaving group (e.g.
halogen, hydroxy, carboxylate, or together form a bi-dentate
carboxylate, or sulphate) and X is NH.sub.3 or mono- or dialkyl
substituted NH.sub.3. Z is a substituted amine, preferably a 5- or
6-membered monocyclic or an 8 10 membered polycylic amine,
especially substituted pyridine or bycylic amine where the amine is
coordinated through the nitrogen atom. Ivanova, N. A. et al.
Russian J. Coord. Chem. (1993) 19(12) 856 863 describe the
synthesis of PtX.sub.2 complexes with piperazine, where X may be Cl
or Br. The synthesized complexes were analyzed with vibrational
spectroscopy for elucidating the piperazine conformation.
Cattalini, L. et al. J. Chem. Soc. Dalton Transactions (1993) 233
236 describe cis and trans PtCl.sub.2 complexes containing two
amine ligands, the amine chosen from pyridine, substituted
pyridine, morpholine, piperidine and dimethylamine. Shionogi &
Co. EP-A-0273315 (1988) disclose cis-PtX.sub.2(NH.sub.3)(Am)
complexes. X maybe Cl, I, nitro or a cyclic moiety and Am is a
substituted C.sub.2-7N and their antitumor activity. Wong et al.
Chem. Rev. (1999) 99(9), 2451 2466 review platinum-based antitumor
drugs mentioning cis-Pt complexes.
In addition, Navarro-Ranniger and co-workers demonstrated that
trans-PtCl.sub.2[NH.sub.2CH(CH.sub.3).sub.2][NH(CH.sub.3).sub.2]
has interesting pharmacological properties.sup.(9) and Natile et.
al. reported that trans-PtCl.sub.2(iminoether).sub.2 is also active
against several human cancer lines.sup.(10). Another example of a
non-classical complex that is in phase 2 of clinical trials is the
trinuclear Pt complex BBR3464 that is a quadruply charged
cation.sup.(11).
The importance of the non-classical platinum compounds stems from
the fact that they were designed to form a spectrum of DNA adducts
that is distinct from that formed by cisplatin and carboplatin and
hence they can circumvent acquired Pt resistance.sup.(12).
Generally, trans-diaminedichloroplatinum(II) analogues have lower
solubility in aqueous solution than their cis counterparts,
resulting in limited bioavailability. One way of increasing the
aqueous solubility is by adding a charge to the complex. Farrell et
al. have put some effort in overcoming the poor water solubility of
compounds of the type
trans-[PtCl.sub.2(NH.sub.3)(Am.sub.1)](Am.sub.1=planar ligand),
while retaining the trans orientation of the NH.sub.3 and the
planar ligand and electroneutralilty of the square-planar entity.
The trans-platinum complex trans-[PtCl(PyAc--N,O)(NH.sub.3)]
(PyAc=pyridin-2-yl acetate, N-donors are trans) and its cis isomer
were synthesized and the trans isomer have shown improved
solubility (ca. 4 5 mmol L.sup.-1) in water, compared to analogous
complexes trans-[PtCl.sub.2(NH.sub.3)(Am.sub.1)](Am.sub.1=planar
ligand).sup.(13). On the other hand, the cationic charges of the
platinum complexes prepared by Farrell et al. and also by Hollis et
al. reside on the metal center and result from the substitution of
one or the anionic chloride ligands by a neutral
ligand.sup.(14).
SUMMARY OF THE INVENTION
The present invention concerns, according to a first of its
aspects, novel Platinum complexes (Pt-complexes) in the trans
configuration having the general formula (I):
[Pt(X)(Y)(Am.sub.1)(Am.sub.2)] (I) wherein: X and Y, which may be
the same or different, represent a halogen, carboxylate, phosphate
or sulphate group; Am.sub.1 represents an amine selected from
ammonia, a primary amine, a secondary amine, a non-planar
heterocyclic aliphatic amine or a heterocyclic aromatic amine; and
Am.sub.2 represents a non-planar heterocyclic aliphatic amine,
wherein the following compounds are excluded
trans-[Pt(piperidine).sub.2Cl.sub.2] and
trans-[Pt(morpholine).sub.2Cl.sub.2].
The Pt-complex of the invention may be in the form of a dimer in
which each monomeric unit is a Pt-complex as defined, bound to the
other Pt-complex, independently, through the Am.sub.1 or through
the Am.sub.2 or through a linker connected to said Am.sub.1 or
Am.sub.2.
According to another aspect, the invention concerns pharmaceutical
compositions comprising a pharmaceutically acceptable carrier and
as an active ingredient a therapeutically effective amount of a Pt
complex of the general formula: [Pt(X)(Y)(Am.sub.1)(Am.sub.2)]
wherein: X and Y, which may be the same or different, represent a
halogen, carboxylate, phosphate or sulphate group; Am.sub.1
represents an amine selected from ammonia, a primary amine, a
secondary amine, a non-planar heterocyclic aliphatic amine or a
heterocyclic aromatic amine; and Am.sub.2 represents a non-planar
heterocyclic aliphatic amine, with the proviso that when said
complex is in a cis configuration, Am.sub.1 and Am.sub.2 cannot
represent simultaneously piperidine.
The invention also concerns a method for achieving a therapeutic
effect, the method comprising administering to a subject in need an
amount of a platinum complex, the amount being sufficient for
achieving said therapeutic effect and the Pt complex comprises the
general formula (I): [Pt(X)(Y)(Am.sub.1)(Am.sub.1)] (I) wherein: X
and Y, which may be the same or different, represent a halogen,
carboxylate, phosphate or sulphate group; Am.sub.1 represents an
amine selected from ammonia, a primary amine, a secondary amine, a
non-planar heterocyclic aliphatic amine or a heterocyclic aromatic
amine; and Am.sub.2 represents a non-planar heterocyclic aliphatic
amine,
with the proviso that when said complex is in a cis configuration,
Am.sub.1 and Am.sub.2 cannot represent simultaneously
piperidine.
According to yet another aspect the invention concerns a platinum
complex of the general formula (I): [Pt(X)(Y)(Am.sub.1)(Am.sub.2)]
(I) wherein: X and Y, which may be the same or different, represent
a halogen, carboxylate, phosphate or sulphate group; Am.sub.1
represents an amine selected from ammonia, a primary amine, a
secondary amine, a non-planar heterocyclic aliphatic amine or a
heterocyclic aromatic amine; and Am.sub.2 represents a non-planar
heterocyclic aliphatic amine;
wherein the following compounds are excluded:
cis-[PtCl.sub.2(quinoline)(piperidine)];
cis-[PtCl.sub.2(piperidine)(pyridine)];
cis-[PtCl.sub.2-(piperidine)(o-CH.sub.3--C.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(piperidine)(p-CH.sub.3--C.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(morpholine)(pyridine)];
cis-[PtCl.sub.2(morpholine)(o-CH.sub.3--C.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(morpholine)(p-CH.sub.3--C.sub.6H.sub.4--NH.sub.2];
cis-[PtCl.sub.2(piperidine)(aniline)];
cis-[PtCl.sub.2(piperidine)(o-CH.sub.3O--C.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(piperidine)(p-C.sub.2H.sub.5OC.sub.6H.sub.4--NH.sub.2)];
cis-[PtCl.sub.2(quinoline)(cyclohexylamine)];
cis-[PtCl.sub.2(quinoline)(morpholine)];
cis-[PtCl.sub.2(quinoline)(piperidine)];
cis-[PtBr.sub.2(piperazine)(piperazine);
cis-[PtCl.sub.2(piperazine)(piperazine)];
cis-[PtCl.sub.2(piperidine)(piperidine)];
cis-PtCl.sub.2(morpholine)(morpholine)];
cis-[PtCl.sub.2(pyrrolidine)(NH.sub.3)];
cis-[PtI.sub.2(pyrrolidine)(NH.sub.3)];
cis-{PtICl(pyrrolidine)(NH.sub.3)];
cis-[PtCl.sub.2(piperidine)(NH.sub.3)];
cis-[PtI.sub.2(piperidine)(NH.sub.3)];
cis-[PtCl.sub.2(piperidone)(NH.sub.3)];
cis-[PtI.sub.2(piperidone)(NH.sub.3)];
cis-[PtICl(piperidone)(NH.sub.3)];
cis-[PtCl.sub.2(3-hydroxypyrrolidine(NH.sub.3)];
cis-[PtI.sub.2(3-hydroxypyrrolidine)(NH.sub.3)],
cis-[PtClI(3-hydroxypyrrolidine)(NH.sub.3),
trans-[Pt(piperidine).sub.2Cl.sub.2] and
trans-[Pt(morpholine).sub.2Cl.sub.2].
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A 1B show uptake by C-26 cancer cells (FIG. 1A) or OV-1063
cancer cells (FIG. 1B) of cisplatin (-?-); transplatin (- -);
trans-[(PtCl.sub.2)(4-picoline)(piperidine)](-^-);trans-[PtCl.sub.2)(4-pi-
coline)(piperazine)].HCl (- -) and
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl
(-.smallcircle.-). The Pt content was determined by Atomic
Absorption Spectroscopy (AAS).
FIGS. 2A 2B show DNA platination levels in C-26 cancer cells (FIG.
2A) or OV-1063 cancer cells (FIG. 2B) of cisplatin (-?-);
transplatin (- -);
trans-[PtCl.sub.2)(4-picoline)(piperidine)](-^-);trans-[PtCl.sub.2)(4-pic-
oline)(piperazine)].HCl (- -) and
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl
(-.smallcircle.-). The Pt content was determined by Atomic
Absorption Spectroscopy (AAS).
FIG. 3 shows the dependence of EtBr fluorescence on different
concentrations of DNA modified by cisplatin (- -); transplatin
(-^-); trans-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl (- -);
trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl (-x-) and
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl (-?-). Data
points were measured in triplicate which varied on average by
.+-.3%.
FIG. 4 shows Caspase-3-activity in OV-1063 cells, which were
treated with IC.sub.50 values of
trans-[PtCl.sub.2(4-picoline)(piperidine)] (6.5 .mu.M,
trans-PtCl.sub.2(4-pic)(pip)]),
trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl (7.5 .mu.M or 6.5
.mu.M, respectively, trans-[PtCl.sub.2(4-pic)(pz)].HCl),
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl (4 .mu.M or
6 .mu.M, respectively, trans-[PtCl.sub.2(NH.sub.3)(pipo-pip)].HCl)
or cisplatin (2 .mu.M or 1 .mu.M, respectively) compared to
untreated (control) cells. Both drug-treated and control cells were
then harvested, lysed, and assayed after the indicated amount of
time, as described in the kit protocol.
FIG. 5 shows the binding curve of Ubiquitin to cisplatin (-?-);
transplatin (- -); trans-[PtCl.sub.2)(NH.sub.3)(piperidine)](-^-);
and trans-[PtCl.sub.2)(NH.sub.3)(piperazine)].HCl (- -).
FIG. 6 shows the antitumor activity of
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl (- -) as
compared to cis-DDP (- -) in female BALB/C mice inoculated with
C-26 colon carcinoma and treated according to the schedule
described herein below.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the surprising finding that
inclusion of a non-planar heterocyclic aliphatic amine ligand into
a Pt-complex, have therapeutic advantages, e.g. in the field of
cancer treatment. The Pt-complex according to the invention
includes at least one non-planar heterocyclic amine ligand, which
is flexible and has a hydrogen bond donor that can interact with
other substances, such as with DNA, to form lesions. The ligand is
also bulky enough to affect the kinetics and the cytotoxicity of
the resulting complex.
Thus, the present invention provides, according to one of its
aspects, a platinum complex (Pt-complex) of the general formula
(I): [Pt(X)(Y)(Am.sub.1)(Am.sub.2)] (I)
wherein: X and Y, which may be the same or different, represent a
halogen, carboxylate, phosphate or sulphate group; Am.sub.1
represents an amine selected from ammonia, a primary amine, a
secondary amine, a non-planar heterocyclic aliphatic amine or a
heterocyclic aromatic amine; and Am.sub.2 represents a non-planar
heterocyclic aliphatic amine,
with the proviso that when said complex is in a cis configuration,
Am.sub.1 and Am.sub.2 cannot represent simultaneously
piperidine.
The term "Pt-complex" as used herein refers in its broadest sense
to any Pt-complex comprising two amine-containing ligands, wherein
at least one ligand is a non-planar heterocyclic aliphatic amine.
These complexes include both the cis and trans regioisomers (with
the proviso that when the complex is in a cis configuration, the
two amine ligands do not represent simultaneously piperidine). The
Pt-complex may include Pt(II) coordinated or Pt(IV) coordinated as
the metal center. In addition, the Pt-complex may be in the form of
a dimer in which each monomeric unit is a Pt-complex as defined
above, bound to the other Pt-complex, independently, through one of
its amine ligands, directly, or via a linker connected to said
Am.sub.1 or Am.sub.2, the two amine ligands may also form together
a cyclic ring, such as a piperizine ring coordinated with each Pt
metal through a nitrogen atom.
According to a preferred embodiment, X and Y are the same or
different and represent chloride or iodide and more preferably, X
and Y both represent a chloride.
According to the invention Am.sub.1 may represent ammonia; a
primary amine such as, without being limited thereto, methylamine,
ethylamine, n-propylamine, isopropylamine, n-butylamine,
n-hexylamine, n-heptylamine or n-nonylamine; a secondary amine such
as, without being limited thereto, dimethylamine, diethylamine,
dipropylamine, dibutylamine; a non-planar heterocyclic aliphatic
amine, such as, without being limited thereto, piperazine,
2-methylpiperazine, piperidine, 2-, 3-, or 4-hydroxypiperidine,
4-piperidino-piperidine, pyrrolidine, 4-(2-hydroxyethyl)piperazine
and 3-aminopyrolidine; or a heterocyclic aromatic amine, such as,
without being limited thereto, pyridine, 2-, 3-, or
4-aminopyridine, 2-, 3-, or 4-picoline, quinoline, 3-, or
4-aminoquinoline, thiazole, imidazole, 3-pyrroline, pyrazine,
2-methylpyrazine, 4-aminoquinaldine.
The Am.sub.2 according to the invention is a non-planar
heterocyclic amine such as, without being limited thereto,
piperazine (referred to herein, at times, by the abbreviation
"pz"), 2-methylpiperazine, piperidine (referred to herein, at
times, by the abbreviation "pip"), 2-, 3-, or 4-hydroxypiperidine,
4-piperidino-piperidine (referred to herein, at times, by the
abbreviation "pip-pip"), pyrrolidine, 4-(2-hydroxyethyl)piperazine
and 3-aminopyrolidine.
As indicated above, the Pt-complexes of the invention refer to all
regioisomers of the complexes having the general formula (I)
identified above. According to one aspect, the Pt-complexes are in
the trans configuration. Specific examples include:
trans-[PtCl.sub.2(NH.sub.3)(piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4-hydroxypiperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4-piperidino-piperidine)];
trans-[PtCl.sub.2(NH.sub.3)(4,4'-bipiperidine)];
trans-[PtCl.sub.2(4-picoline)(piperidine)];
trans-[PtCl.sub.2(piperidine).sub.2];
trans-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl;
trans-[ptcl.sub.2(isopropylamine)(piperazine)].HCl;
trans-[PtCl.sub.2(n-butylamine)(piperazine)].HCl;
trans-[PtCl.sub.2(n-nonylamine)(piperazine)].HCl
trans-[PtCl.sub.2(piperidine)(piperazine)].HCl;
trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl;
trans-[PtCl.sub.2(piperazine)(piperazine)].HCl;
trans-[PtCl.sub.2(NH.sub.3)[4-(2-hydroxyethyl)piperazine)].HCl;
According to yet another aspect, the complexes are in a cis
configuration. Specific cis isomers include, without being limited
thereto, cis-[PtCl.sub.2(NH.sub.3)(piperidine)] or
cis-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl.
As indicated above, the non-planar heterocyclic amine ligand is
flexible and has a hydrogen bond donor that can interact with the
DNA to form lesions and is bulky enough to affect the kinetics and
the cytotoxicity of the resulting complex. In addition, some of the
amine ligands such as piperazine confer the complex with a positive
charge, thus ensuring adequate aqueous solubility and rapid
interaction of the complex with polyanionic molecules, such as the
DNA.
The complex may also be in the form of a dimer. Accordingly, two
Pt-complexes are associated via a valance bond, a cyclic ring
formed between the amine substituent of each Pt-complex (e.g.
forming together a piperazine ring) or by a linker connected to the
Am.sub.1 or the Am.sub.2 ligands of each complex. Non-limiting
examples of linkers include short polyethyleneglycol chains (PEG),
short diaminoalkanes (e.g. 1,6-diaminohexane, 1,8-diaminooctane). A
specific example for a linker is 4,7,10-trioxa-1,13-tridecane chain
and one specific dimer in which the two Pt-complexes are associated
by this linker is Bis-[{trans,
trans-(PtCl.sub.2piperazine).sub.2}(4,7,10-trioxa-1,13-tridecanediamine)]-
.2HCl.
The invention also concerns pharmaceutical compositions comprising
a pharmaceutically acceptable carrier and as an active ingredient a
therapeutically effective amount of the Pt-complex of the invention
as defined above.
The Pt-complex of the present invention is administered and dosed
in accordance with good medical practice, taking into account the
clinical condition of the individual patient, the site and method
of administration, scheduling of administration, patient age, sex,
body weight and other factors known to medical practitioners. The
therapeutically "effective amount" for purposes herein is thus
determined by such considerations as are known in the art. The
amount must be effective to achieve improvement including, but not
limited to, improved survival rate or more rapid recovery from a
disease state treated with the active ingredient of the invention,
or improvement or elimination of symptoms associated with the
disease state and other indicators as are selected as appropriate
measures by those skilled in the art.
The effective amount is typically determined in appropriately
designed clinical trials (dose range studies) and the person versed
in the art will know how to properly conduct such trials in order
to determine the effective amount. As generally known, an effective
amount depends on a variety of factors including the affinity of
the Pt-complex to, for example, DNA, to form a Pt-DNA adduct, the
Pt-complex's distribution profile within the body, a variety of
pharmacological parameters such as half life in the body, on
undesired side effects, if any, on factors such as age and gender,
etc.
Many modes of administration may be employed for the delivery of
Pt-complex, and these will necessitate the use of different
carriers, adjuvants, elixirs, and the like, as known in the
art.
Evidently, the pharmaceutically acceptable carriers employed
according to the invention generally refer to inert, non-toxic
solid or liquid fillers, diluents or encapsulating material to the
extent that they do not hinder or interfere with the therapeutic
effect desired of the Pt complex and do not react with the
Pt-complex of the invention.
The Pt-complex can be administered orally, subcutaneously or
parenterally including intravenous, intraarterial, intramuscular,
intraperitoneally and intranasal administration as well as
intrathecal and by infusion techniques. Further, the Pt-complex can
be suspended in chlorofluorocarbon or hydrofluorocarbon propellants
for delivery via inhaler to the lungs. Alternatively, the
Pt-complex can be formulated in a matrix (lactose, etc.) or carrier
(e.g., liposomes, etc.), which will allow delivery either orally,
sublingually or by suppository.
The doses may be single doses or multiple doses over a period of
several days. The treatment generally has a length proportional to
the length of the disease process and active ingredient
effectiveness and the patient species being treated. Further, the
administration of Pt-complex of the present invention can be
intermittent, or at a gradual, or continuous, constant or
controlled rate to a patient. The host or patient for the
therapeutic treatment using the platinum compounds described herein
generally are mammalian, such as humans, dogs, and rodents, and so
forth.
When administering the Pt-complex of the invention parenterally, it
will generally be formulated in a unit dosage injectable form
(solution, suspension, emulsion). The pharmaceutical formulation
suitable for injection includes sterile aqueous solutions or
dispersions and sterile powders for reconstitution into sterile
injectable solutions or dispersions. The carrier can be a solvent
or dispersing medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, lipid polyethylene
glycol and the like), suitable mixtures thereof and vegetable oils.
Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil,
soybean oil, corn oil, sunflower oil, or peanut oil and ester, such
as isopropyl myristate, may also be used as solvent systems for the
Pt-complex of the invention.
Additionally, various additives which enhance the stability,
sterility and isotonicity of the Pt-complex containing compositions
of the invention, including antimicrobial preservatives,
antioxidants and buffers can be added. Prevention of the action of
microorganisms can be ensured by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid and the like.
The pharmaceutical composition of the invention may also be
administered orally to the subject in need. Conventional methods
such as administering the active compound in tablets, suspensions,
solutions, emulsions, capsules, powders, syrops and the like are
usable. Known techniques which deliver it orally or intravenously
and retain the biological activity thereof are preferred.
According to one preferred embodiment, the Pt-complex of the
invention is is entrapped by or loaded onto a liposome. The term
"liposome" as used herein includes all spheres or vesicles of
liposome-forming lipids that may spontaneously or non-spontaneously
vesiculate, for example, phospholipids which are glycerides where
at least one acyl group is replaced by a complex phosphoric acid
ester.
The term "loaded" or "entrapped" means either entrapped within the
interior of the liposome, exposed or present at the surface of the
liposome, embedded in the liposome's membrane.
The liposomes according to the invention may be formed from any
known liposome forming lipids. As used herein, the term
"liposome-forming lipid" denotes any physiologically acceptable
amphipathic substance that contains groups with characteristically
different properties, e.g. both hydrophilic and hydrophobic
properties or a mixture of such molecules, and which upon
dispersion thereof in an aqueous medium form liposomal vesicles.
The liposomes may be comprised of a single amphipathic substance or
from a mixture of such substances.
The amphipathic substance includes, inter alia, phospholipids,
sphingolipids, glycolipids, such as cerebrosides and gangliosides,
PEGylated lipids, and sterols, such as cholesterol and others. Any
commonly known liposome-forming lipids are suitable for use by the
method of the present invention. The source of the lipid or its
method of synthesis is not critical: any naturally occurring lipid,
with and without modification, or a synthetic phosphatide can be
used.
Preferred liposome-forming amphipathic substances are natural,
semi-synthetic or fully synthetic, molecules; negatively or
positively charged lipids, phospholipids or sphingolipids,
optionally combined with a sterol, such as cholesterol; and/or with
lipopolymers, such as PEGylated lipids.
The liposomes employed by the invention can be "tailored" to the
requirements of any specific reservoir including various biological
fluids, which maintain their stability without aggregation or
chromatographic separation, and thereby remain well dispersed and
suspended in the injected fluid. The fluidity in situ changes due
to the composition, temperature, salinity, bivalent ions and
presence of proteins. The liposomes can be used with or without any
other solvent or surfactant.
A preferred phospholipid combination according to the invention
includes a mixture of (HSPC):cholesterol:PEG.sup.2000-DSPE (HSPC
referring to hydrogenated soybean phosphatidylcholine while
PEG.sup.2000-DSPE refers to Di-stearoyl-phosphatidyl-ethanolamine
to which PEG.sup.2000 is bound to the head group) or alternatively,
diacylglycol PEG (having two stearoyl acyl chains) or
cholesterol-PEG.
The composition of the invention is intended for achieving a
therapeutic effect, the therapeutic effect involving the formation
of an adduct between the Pt complex of the invention and a nucleic
acid molecule such as a DNA. The therapeutic effect may comprise
inhibition of undesired cell proliferation or for induction of
apoptosis of undesired cells.
Thus, the composition of the present invention may be used for the
treatment or prevention of a disease state, the disease state being
associated with undesired cell proliferation. The term "treatment
or prevention" as used herein refers to the administering of a
therapeutic amount of the composition of the invention which is
effective to ameliorate undesired symptoms associated with the
disease state, to prevent the manifestation of such symptoms before
they occur, to slow down the progression of the disease (As may be
evident from rate of proliferation of a diseased tissue), slow down
the deterioration of symptoms, to enhance the onset of remission
period, slow down the irreversible damage caused in the progressive
chronic stage of the disease (for example, in autoimmune diseases),
to delay the onset of said progressive stage, to lessen the
severity or cure the disease, to improve survival rate or to
achieve a more rapid recovery, or to prevent the disease form
occurring or a combination of two or more of the above.
Thus, the instant invention also concerns a method for achieving a
therapeutic effect, the method comprising administering to a
subject in need an amount of a platinum complex according to the
invention, the amount being sufficient for achieving said
therapeutic effect. According to one embodiment, the Pt-complex of
the invention is used as an anti-cancer agent.
The invention will now be further explained by the following
non-limiting examples. While the foregoing description describes in
detail only a few specific embodiments of the invention, it will be
understood by those skilled in the art that the invention is not
limited thereto and that other variations in form and details may
be possible without departing from the scope and spirit of the
invention as defined by the claims, which are to be read as
included within the disclosure of the specification.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
EXAMPLE 1
Chemical Synthesis
(1) Synthesis of Piperidine-containing Pt Complexes
General Procedure for Preparing trans-[PtCl.sub.2(NH.sub.3)(R)]
In the following description R refers to any one of the following
piperidine derivatives: piperidine, 4-hydroxypiperidine,
4-piperidino-piperidine, 1,4'-bispiperidine. For further reference,
the derivatives obtained will be identified by a reference number
appearing in brackets.
Cis-diamminedichloroplatinum(II) (300 mg, 1 mmol) was suspended in
30 mL of double distilled water DDW. Two equivalents (eq.) of the
piperidine derivative were added, and the suspension was heated to
85.degree. C. for 3 h. During this time the yellow suspension
turned to a colorless clear solution (in some cases a black
precipitate was formed). The reaction mixture was cooled to r.t.,
filtered, and 1 mL of concentrated HCl was added dropwise. The
temperature was elevated to 90.degree. C. for 6 h, during which the
yellow product, trans-[PtCl.sub.2(NH.sub.3)(piperidine
derivative)], precipitated. The reaction mixture was allowed to
stand at r.t. for 4 h, after which the yellow product was collected
by filtration and washed with 40 mL of DDW, 10 mL of EtOH, and 40
mL of diethyl ether.
Trans-[PtCl.sub.2(NH.sub.3)(piperidine)] (1): Yield 86.9%. Anal.
(C.sub.5H.sub.14Cl.sub.2N.sub.2Pt) C,H,N. .sup.195Pt NMR(.delta.,
DMF): -2167 ppm.
Trans-[PtCl.sub.2(NH.sub.3)(4-hydroxypiperidine)] (2): Yield 80.0%.
Anal. (C.sub.5H.sub.14Cl.sub.2N.sub.2OPt) C,H,N. .sup.195Pt
NMR(.delta., DMF): -2172 ppm.
Trans-[PtCl.sub.2(NH.sub.3)(4-piperidino-piperidine)] (3): Yield
78.5%. Anal. (C.sub.10H.sub.24Cl.sub.3N.sub.3Pt) C,H,N. .sup.195Pt
NMR(.delta., H.sub.2O): -2170 ppm.
Trans-[PtCl.sub.2(NH.sub.3)(4,4'-bipiperidine)] (4): Yield 85.9%.
Anal. (C.sub.10H.sub.24Cl.sub.3N.sub.3Pt) C,H,N. .sup.195Pt
NMR(.delta., H.sub.2O): -2175 ppm.
Procedure for Preparing trans-[PtCl.sub.2(4-picoline)(piperidine)]
(5)
K.sub.2PtCl.sub.4 (200 mg, 0.482 mmol) was dissolved in 30 mL of
DDW. 4-Picoline (2.5 eq., 117.3 .mu.L; 1.2 mmol) was added and the
mixture stirred overnight at r.t. The yellow precipitate,
cis-[PtCl.sub.2(4-picoline).sub.2] [.sup.195Pt NMR(DMF)=-1964 ppm],
was collected by filtration and washed with 50 mL of DDW and 40 mL
of diethyl ether. Cis-[PtCl.sub.2(4-picoline).sub.2] (226 mg, 0.5
mmol) was suspended in 40 mL of DDW with 2 eq. of piperidine (99
.mu.L, 1 mmol), and the suspension was heated to 80.degree. C. for
3 h. The solution turned clear and colorless with some formation of
a black precipitate The reaction mixture was allowed to cool to
r.t. and the precipitated material was filtered off. To the
colorless filtrate, 1 mL of concentrated HCl was added and the
mixture was heated to 90.degree. C. The heating was maintained for
6 h during which a yellow precipitate was formed. The reaction
mixture was allowed to cool to r.t., and the precipitate (180 mg)
was collected and washed with 50 mL of DDW, 10 mL of EtOH, and 30
mL of diethylether.
Yield: 81%. Anal. (C.sub.11H.sub.18C.sub.12N.sub.2Pt): C,H,N.
.sup.195Pt NMR(.delta., DMF): -2087 ppm.
Procedure for Preparing Trans-[PtCl.sub.2(piperidine).sub.2]
(6)
K.sub.2PtCl.sub.4 (415 mg, 1 mmol) was dissolved in 50 mL of DDW to
which (1.330 gr, 8 mmol) KI were added and the red solution was
stirred at r.t. for 20 minutes. To the stirred solution (202 .mu.l,
2 mmol) of piperidine were added slowly. After 1 hr of stirring at
r.t. the yellow precipitate was collected and washed with 50 mL of
DDW and then with 50 mL of ether. Cis-[PtI.sub.2(Pip).sub.2] (300
mgs, 0.484 mmol) was taken up in 20 mL DMF and, 164.5 mgs (0.97
mmol) of AgNO.sub.3 and (98 .mu.l, 1.94 mmol) were added and the
mixture was stirred overnight at r.t. After filtering off the
precipitate the solution was evaporated to dryness. To the gum 20
mL of DDW were added and stirred for 30 minutes. The non-soluble
materials were filtered and 2 mL of concentrated HCl were added.
The acidified solution was wormed to 90.degree. C. for 5 hrs. After
cooling to r.t. the yellow precipitate was collected and washed
with 50 mL DDW and 40 mL ether.
Trans-[PtCl.sub.2(piperidine).sub.2] (6): Yield: 91%. Anal.
(C.sub.10H.sub.22Cl.sub.2N.sub.2Pt): C,H,N. .sup.195Pt NMR(.delta.,
DMF): -2080 ppm.
Procedure for Preparing cis-[PtCl.sub.2(NH.sub.3)(piperidine)]
(7)
K.sub.2PtCl.sub.4 (415 mg, 1 mmol) of was dissolved in 50 mL of
DDW, and 8 eq. KI (1.328 g, 8 mmol) were added. The mixture was
stirred at r.t. for 15 min., and then 2 eq. of piperidine (198
.mu.L, 2 mmol) were added slowly. The mixture was stirred for 1 h
at r.t., during which a yellow precipitate was formed. The
precipitate was collected and washed thoroughly with 50 mL of DDW,
and with 20 mL of a (1:1) acetone: diethyl ether mixture. After
drying, the yellow precipitate (500 mg, 0.8 mmol) was suspended in
a mixture of 20 mL of DDW and 40 mL of ethanol, to which 1 mL of
perchloric acid (70%) was added. The suspension was stirred at r.t.
for 8 days. During this period, the yellow precipitate turned to
brown. The brown precipitate was collected by filtration and washed
with 40 mL of DDW and 20 mL of acetone:diethylether (1:1). After
drying, the precipitate was re-suspended in 20 mL of DDW, 0.5 mL of
25% NH.sub.4OH was added dropwise, and the mixture was vigorously
stirred for 24 h, during which the brown-colored precipitate turned
to yellow. The yellow precipitate was collected and washed
thoroughly with 50 mL of DDW, and 10 mL of acetone-diethyl ether
and dried by continuous suction. The product was characterized as
the mixed cis-amine-piperidine-diiodoplatinum(II) [.sup.195Pt
NMR(.delta., DMF)=-3260 ppm].
Cis-amine-piperidine-diiodoplatinum(II) (300 mg, 0.54 mmol) was
suspended in 20 mL DDW, and 2 eq of AgNO.sub.3 (184.9 mg, 1.08
mmol) were added. The suspension was vigorously stirred in the dark
for 24 h. The AgI precipitate was filtered off, and the aqueous
filtrate was transferred into a 50-mL vessel, to which 0.5 g of KCl
were added. The colorless solution turned yellowish, and the
dichloro-diamineplatinum(II) product started precipitating. After 4
h at r.t. the yellowish precipitate (260 mg) was collected and
washed thoroughly with 50 mL of DDW and dried by washing with 100
mL of diethyl ether.
Overall yield of cis-[PtCl.sub.2(NH.sub.3)(piperidine)] (7): 69%.
Anal. (C.sub.5H.sub.14Cl.sub.2N.sub.2Pt) C,H,N. .sup.195Pt
NMR(.delta., DMF): -2159 ppm.
(2) Synthesis of Pt Piperazine Complexes
One aim of the research presented herein was to design and prepare
platinum complexes that are water-soluble, react rapidly with DNA
and are able to form adducts with DNA that are different from those
formed by cisplatin and transplatin. This led to the design and
synthesis of several additional trans-Pt derivatives with the
piperazine as a ligand. This ligand was chosen since it would
confer positive charge to the complex and thus would ensure
adequate aqueous solubility and rapid interaction with the
polyanionic DNA; it is a non-planar heterocyclic amine ligand that
is flexible and has a hydrogen bond donor that can interact with
the DNA to form a lesion; and it is bulky enough to affect kinetics
and cytotoxicity.
Trans-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl (8)
Cis-diammine-dichloroplatinum(II), (300 mg, 1 mmol) was dissolved
in 30 mL of DMF and 2 eq. (372.52 mg, 2 mmol) of tert-butyl
1-piperazine carboxylate and 2 eq. (339.76 mg, 2 mmol) of
AgNO.sub.3 were added simultaneously with stirring. Stirring
continued in the dark for 24 h at room temperature. The precipitate
was filtered off through a celite sinter glass and the filtrate was
evaporated to dryness under reduced pressure. The resulting gum was
dissolved in 30 mL of DDW and 2 mL of concentrated HCl were added
and the reaction mixture was stirred at r.t. for 24 h. The colored
precipitates were removed and the solution was heated to 85
90.degree. C. for 60 min. After cooling to room temperature the
reaction mixture was filtered and the filtrate was chilled to
0.degree. C. for 72 h. The yellow precipitate was filtered and
washed with 10 mL of ice-cold DDW and 30 mL of diethyl ether. After
drying, the yellow product (300 mg) was characterized as the
hydrochloride salt of the desired
trans-ammine-piperazine-dichloroplatinum(II) (8)
Trans-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl (8) Yield: 74%. Anal.
Calcd. for C.sub.4H.sub.14Cl.sub.3N.sub.3Pt.H.sub.2O: C, 11.34%; H,
3.81%; N, 9.92%. Found: C, 11.27%; H, 3.56%; N, 9.86%.
.sup.195Pt-NMR(.delta., H.sub.2O): -2177 ppm.
General Procedure for Preparing
trans-[PtCl.sub.2(Am1)(piperazine)].HCl
In the following description Am1 refers to any one of the following
amines: n-butyl amine, isopropyl amine, 4-picoline, piperidine,
piperazine.
Synthesis of the intermediate cis-[PtI.sub.2(tert-butyl
1-piperazine carboxylate).sub.2]: Potassium tetrachloroplatinate (1
g, 2.4 mmol) was dissolved in 40 mL of DDW and 8 eq. (3.2 g, 19.27
mmol) of KI were added. The mixture was stirred at r.t. for 15 min.
Two eq. (0.9 g, 4.8 mmol) tert-butyl 1-piperazine carboxylate were
added and the mixture was vigorously stirred for 1 h at r.t.
Throughout this period of time the desired
diiododiamineplatinum(II) precipitated. The yellow precipitate was
collected by filtration, washed with 50 mL of DDW and dried by
suction.
Cis-[PtI.sub.2(tert-butyl 1-piperazine carboxylate).sub.2: Yield:
89%, .sup.195Pt-NMR(.delta., DMF): -3264 ppm.
Cis-[PtI.sub.2(tert-butyl 1-piperazine carboxylate).sub.2 (411 mg,
0.5 mmol) was dissolved in the dark in 15 mL of DMF and 2 equiv
(169.88 mg, 1 mmol) of AgNO.sub.3 were added simultaneously with 2
eq. of the corresponding amine (98.83 .mu.L of n-butylamine, 85.17
.mu.L of isopropylamine, 97.4 .mu.L of 4-picoline, 99 .mu.L of
piperidine, or 186 mg tert-butyl 1-piperazine carboxylate).
Stirring continued in the dark for 24 h at r.t. The precipitate was
flittered off through a celite sinter glass. The filtrate was
evaporated to dryness under reduced pressure. The resulting gum was
dissolved in 30 mL of DDW and 2 mL of concentrated hydrochloric
acid were added and the reaction mixture was stirred at room
temperature for 24 h. The colored precipitates were removed and the
solution was heated to 85 90.degree. C. for 60 min. After cooling
to room temperature the reaction mixture was filtrated and the
filtrate was chilled to 0.degree. C. for 24 hours. The yellow
precipitate was filtered and washed with 20 mL of ice cooled DDW
and 30 mL of diethyether. After drying, the yellow products were
characterized as the hydrochloride salts of the desired
trans-diamine-dichloroplatinum(II) complex.
Trans-[PtCl.sub.2(isopropylamine)(piperazine)].HCl (9): Yield 71%,
.sup.195Pt-NMR(.delta., H.sub.2O): -2226 ppm.
Trans-[PtCl.sub.2(n-butylamine)(piperazine)].HCl (10): Yield 67%,
.sup.195Pt-NMR(.delta., H.sub.2O): -2221 ppm.
Trans-[PtCl.sub.2(n-nonylamine)(piperazine)].HCl (11): Yield 77%,
.sup.195Pt-NMR(.delta., H.sub.2O): -2236 ppm.
25.06%, H: 3.82%, N: 8.55%, .sup.195Pt-NMR(.delta., H.sub.2O):
-2086 ppm.
Trans-[PtCl.sub.2(piperidine)(piperazine)].HCl (12): Yield 56%,
.sup.195Pt-NMR(.delta., H.sub.2O): -2230 ppm.
Trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl (13): Yield 61.2%,
Anal. Calc. For C.sub.10H.sub.18Cl.sub.3N.sub.3Pt: C: 24.99%, H:
3.56%, N: 8.74%, Found: C: 25.06%, H: 3.82%, N: 8.55%,
Trans-[PtCl.sub.2(piperazine)(piperazine)].HCl (14): Yield 83%,
.sup.195Pt-NMR(.delta., H.sub.2O): -2238 ppm.
Trans-[PtCl.sub.2(NH.sub.3)[4-(2-hydroxyethyl)piperazine)].HCl
(15): Yield 83%, .sup.195Pt-NMR(.delta., H.sub.2O): -2238 ppm.
Procedure for the Preparation of
cis-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl (16)
Tetraphenylphosphonium trichloro-monoammine-platinum(II) (300 mg,
0.45 mmol) were dissolved in 10 mL of 1:1 acetone/DDW mixture. To
the orange-colored solution 1 equiv (77.53 mg, 0.45 mmol)
tert-butyl 1-piperazine carboxylate was added. The mixture stirred
in a closed vessel at r.t. for 7 days. After evaporating the
solution to dryness under reduced pressure the yellow solid was
taken in 10 mL of absolute ethanol and 0.5 mL of concentrated
hydrochloric acid was added and the mixture was allowed to stand
for overnight. The yellow precipitate was collected by filtration
and washed with 10 mL of ethanol.
Cis-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl: Yield: 58%,
.sup.195Pt-NMR(.delta., H.sub.2O): -2187 ppm.
EXAMPLE 2
Biological Assays
Cell Cultures
A human ovarian carcinoma cell line (OV-1063), established at the
Hadassah University hospital and human colon carcinoma cell line
(C-26) were maintained in RPMI-1640 medium supplemented with 10%
FCS, antibiotics and glutamine. All culture medium components were
purchased from Biological Industries (Beit-HaEmek, Israel). Both
cell lines were maintained at 37.degree. C. in a water-jacketed
CO.sub.2 incubator.
Further, three pairs of cisplatin sensitive and resistant cancer
cell lines (A2780/A2780cisR, 41M,/41McisR and CH1/CH1cisR) were
employed.sup.(15). These pairs of cell lines were selected on the
basis of encompassing all of the known major mechanisms of
resistance to cisplatin: 41McisR being resistant primarily through
reduced drug transport.sup.(16), CH1cisR through enhanced DNA
repair/tolerance.sup.(17) and A27780cisR through a combination of
decreased uptake, enhanced DNA repair/tolerance and elevated GSH
levels.sup.(18).
Drugs
Cisplatin and transplatin were supplied by (Sigma, St Louis, Mo.,
USA). All drugs were dissolved in normal saline immediately before
the experiments.
Methylene Blue Assay of Cell Survival
Cytotoxicity of the synthesized complexes was tested by the
methylene blue (MB) staining assay.sup.(19). A fixed number of
exponentially growing cells in 200 .mu.l medium were plated in
96-microwell, flat-bottomed plates. For each of the complexes
tested, 4 well were used. Following 24 hr in culture, 20 .mu.l of
different concentration of the complexes were added to each well
containing untreated cells. Normal saline was added to the
controls. Cells were exposed to complexes for 4, 24, or 72 hr. At
the end of exposure for a fixed time interval, the treated cells as
well as parallel control cells were washed and incubation was
continued in fresh medium until termination of the experiment.
Following 72 hr of growth, cells were fixed by adding 50 .mu.l of
2.5% of glutaraldehyde to each well for 15 min. Fixed cells were
rinsed 10 times with fresh de-ionized water and once with borate
buffer (0.1 M, pH=8.5), dried and stained with MB (100 .mu.l of 1%
solution in 0.1 M borate buffer, pH=8.5) for 1 hr at room
temperature. Stained cells were rinsed thoroughly with de-ionized
water to remove any non-cell-bound die and then dried. The MB bound
to the fixed cells was extracted by incubation at 37.degree. C.
with 200 .mu.l of 0.1N HCl for 1 hr, and the net optical density
(OD) of the die in each well was determined by a plate
spectrophotometer (Labsystems Multyskan BICHROMATIC, Finland) at
620 nm.
The advantage of the MB method with 96-microwell plates is the
possibility of running a wide range of experiments on the rate of
cell proliferation and survival with a large number of data points,
where cells are grown in the same plate and assayed exactly in the
same conditions for different experimental complexes. The validity
of the MB assay for evaluating cell survival is supported by the
high correlation between the MB colorimetric assay and
colony-forming units assay results.sup.(20).
Microculture Tetrazolium (MTS) Assay and Cell Survival
Cytotoxicity of the synthesized complexes was also tested by the
MTS method.sup.(21). Accordingly, the compounds were incubated for
24 hours with the corresponding cell lines and the cell survival in
compound-treated cultures was evaluated.
Platinum Complex Intracellular Accumulation Measurement
Cells were seeded for 48 hr before one of the complexes was added
to the culture medium. After 24 hr of exposure, the complexes were
removed and the cells washed twice with ice-cold PBS and pelleted.
The cells (1*10.sup.6) were dried and mineralized by heating for 10
min in 65% HNO.sub.3 (BDH, England).sup.(22). Samples were
dissolved in de-ionized water and each sample was measured at two
different dilutions by flameless Zeeman atomic absorption
spectrometer (FAAS). The calibration curves included 5 standards of
K.sub.2PtCl.sub.4 stock solution with concentrations ranging from
50 to 250 ng platinum per ml. Platinum content was expressed as
picomoles platinum per 1*10.sup.6 cells.
Determination of Pt-DNA Adducts by FAAS
Cells were seeded for 48 hr before one of the drugs was added to
the culture medium. After 24 hr of exposure, the complexes were
removed and the cells washed twice with ice-cold PBS and pelleted.
DNA from platinum-containing material (2*10.sup.6 cells) was
extracted from the cell pellet by QIAamp DNA Blood Kit (QIAGEN,
Germany) according to the manufacture instructions. DNA yield was
determined by measuring the concentration of DNA in the eluate by
absorbance at 260 nm. The DNA isolated from each sample averaged
50.+-.10 .mu.g/ml. Purity is determined by calculating the ratio of
absorbance at 260 nm to 280 nm; the grade of purification of DNA
was on average 95%.
Determination of Pt-DNA Adducts by EtBr Fluorescence
A plasmid (4.8 kbp) containing a gene coding for human growth
hormone, plasmid pS16-hGH, was prepared as previously
described.sup.(23). The freshly prepared DNA was analyzed by
agarose gel (1%) electrophoresis using post-staining with SYBR
Green I fluorescent dye (Molecular Probes, Eugene, Oreg.).
Quantitative analysis of supercoiled plasmid.sup.(24) was performed
and showed that the plasmid DNA was 85 90% in a supercoiled form.
UV-spectroscopy showed no presence of protein or RNA contamination
in any of the DNA batches. The ratio of absorbance at 260 nm to
that at 280 nm was always between 1.8 and 1.9.
DNA were modified by the platinum complexes in 10 mM NaClO.sub.4
(pH 7.0) at 37.degree. C. in the dark for 24 hr. Measurements of
EtBr fluorescence were performed on an LS50B luminescence
spectrometer (Perkin Elmer, Norwalk, Conn.). Fluorescence
measurements of DNA modified by platinum in the presence of EtBr
were performed using the excitation wavelength of 546 nm (slit 10
run) and emission wavelength of 590 nm (slit 10 nm) at 25.degree.
C. The concentrations were 0.01 mg/ml for DNA and 0.04 mg/ml for
EtBr, which corresponded to the saturation of all intercalation
sites of EtBr in DNA.
Assessment of Apoptosis
Apoptosis was assessed by two approaches:
(1) By staining of the C-26 and OV-1063 cells with Merocyanine 540
(MC 540) (Molecular probes, Oregon, USA) and 4',
6-diamidino-2-phenylindone dihydrochloride (DAPI) (Molecular
probes, Oregon, USA). This assay is based on the observation that
soon after the initiation of apoptosis, phosphatidylserine (PS)
translocates from the inner face of the plasma membrane to the cell
surface. At this point, PS can be easily detected by staining with
MC 540, which has a strong affinity to PS.sup.(25). Chromatin
condensation was assessed by staining with DAPI, which
preferentially stains double stranded DNA. In the following
experiments samples containing 5*10.sup.5 cells were cultured on
6-well plates covered with a glass coverslip. After treatment of
cells with IC.sub.50 of the complexes, cells were washed with PBS
and incubated for 2 min in the dark in 500 .mu.l of PBS containing
2.5 .mu.l of MC 540 (1 mg/ml). After that cells were washed with
PBS, fixated with 4% formaldehyde and stained with 300 .mu.l DAPI
(3 .mu.M). Thereafter, glass coverslip was placed on a glass slide
and photographed using a fluorescence confocal microscope.
(2) By the EnzChektm Caspase-3 Assay Kit (Molecular probes, Eugene,
Oreg.). This Kit allows the detection of apoptosis by assaying for
increases in caspase-3 and other DEVD-specific protease activities
(e.g., caspase-7). The basis for the assay is rodamine 110
bis-(N-CBZ-aspartil-L-glutarnyl-L-valyl-aspartic acid amide)
(Z-DEVD-R110). This substrate is a bisamide derivative of rhodamine
110(R110) containing DEVD peptides covalently linked to each of
R110's amino groups. Upon enzymatic cleavage, the non-fluorescent
bisamide substrate converted to the fluorescent R110, which can be
quantified by fluorescence microplate reader using excitation at
485.+-.10 nm and emission detection at 535.+-.10 nm.
Briefly, C-26 and OV-1063 cells were treated with IC50 of
trans-[PtCl.sub.2(4-picoline)(piperidine)] (5) (4.5 .mu.M and 6.5
.mu.M, respectively) and of
trans-[PtCl.sub.2(4-picoline)(piperazine).HCl] (12) (5 .mu.M and
7.5 .mu.M, respectively) for 5 or 16 hr. Both "induced" and
"control" cells were then harvested and lysed. Enzyme reactions
were performed in 96-well plates with 50 .mu.g of cytosolic
proteins (55 minutes of incubation) and with a final concentration
of 25 .mu.M Z-DEVD-R110 substrate as described in the kit
protocol.
In Vivo Toxicity and Anti-tumor Effect
The trans-platinum(II) derivates,
trans-[PtCl.sub.2(NH.sub.3)(4-piperidino-piperidine)] (3) was
evaluated for its toxicity and antitumor efficacy as compared to
cis-DDP.
Toxicity
Toxicity of the
trans-[PtCl.sub.2(NH.sub.3)(4-piperidino-piperidine)] was evaluated
on 8 week-old female BALB/C mice and compared to cis-DDP. This
novel complex and cis-DDP at different concentrations were injected
i.v., three times at weekly intervals, and animal weight and
survival were evaluated.
Antitumor Efficacy
Female BALB/C mice (in the weight range of 17 20 g) were injected
i.p. with 1*10.sup.6 C-26 colon carcinomas. The viability of these
cells was >90% by trypan blue exclusion.
The therapeutic efficacy of trans-[PtCl.sub.2
(NH.sub.3)(piperidino-piperidine)].HCl (3) was studied and compared
to cis-DDP. Treatment began at day 3 after tumor inoculation and
was repeated twice for a total of three injections at weekly
intervals.
Results
Solubility of the Pt Complexes
The low solubility of the neutral diaminedichloro platinum(II)
compounds, that results in poor bioavailability, was one of the
reasons for the design and synthesis of the positively charged
complexes of the general formula [PtCl.sub.2(Am)(pz)].HCl. The
Compounds presented herein were shown to be more soluble than their
neutral counterparts having solubilities in the range of 20 mM
compared with 6.3 mM for cisplatin and 0.8 mM for transplatin. For
example, trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl (13)
exhibited solubility in DDW (at 37.degree. C.) 7.5 mg/ml (18.0
mM).
Biological Activity
In Vitro Growth Inhibition
In order to assess the anti-tumor activity of the synthesized trans
and cis complexes C-26 and OV-1063 cells were incubated for 4, 24,
or 72 hrs with these complexes. MB cytotoxicity assay revealed that
replacing one (NH.sub.3) or both of the transplatin enhances
significantly (by more than fourfold) the cytotoxicity of the new
trans-PtCl.sub.2 compounds in both C-26 and OV-1063 cancer cell
lines (Table 1).
TABLE-US-00001 TABLE 1 IC.sub.50 of complexes against C-26 cells
and OV-1063 cells as compared to cisplatin and transplatin C-26
cells OV-1063 cells Complex IC.sub.50, .mu.M IC.sub.50, .mu.M
IC.sub.50, .mu.M IC.sub.50, .mu.M IC.sub.50, .mu.M IC.sub.50, .mu.M
No. 4 h 24 h 72 h 4 h 24 h 72 h Cisplatin 1.5 .+-. 1.3 0.6 .+-. 0.1
0.2 .+-. 0.2 2.0 .+-. 0.5 0.7 .+-. 0.2 0.5 .+-. 0.2 Transplatin
64.0 .+-. 2.0 46.0 .+-. 3.1 41.0 .+-. 1.7 81.0 .+-. 4.3 73.0 .+-.
5.1 52.0 .+-. 3.2 trans-[PtCl.sub.2(4-picoline)(piperidine)] 5 4.5
.+-. 0.7 2.5 .+-. 0.7 1.75 .+-. 1.0 6.5 .+-. 0.7 6.0 .+-. 1.4 4.5
.+-. 2.1 trans-[PtCl.sub.2(NH.sub.3)(4-picoline)] 12.0 .+-. 1.2
11.0 .+-. 2.0 11.0 .+-. 1.1 18.0 .+-. 3.7 16.0 .+-. 3.5 14.0 .+-.
2.3 trans-[PtCl.sub.2(NH.sub.3)(piperidine)] 1 8.5 .+-. 2.0 7.7
.+-. 2.0 7.0 .+-. 2.5 11.0 .+-. 2.0 9.5 .+-. 1.7 8.7 .+-. 1.4
trans-[PtCl.sub.2(4-picoline) 13 5.5 .+-. 1.0 4.5 .+-. 0.75 3.5
.+-. 0.8 7.4 .+-. 1.5 6.0 .+-. 1.0 5.1 .+-. 0.9 (piperazine)].HCl
cis-[PtCl.sub.2(NH.sub.3)(4-picoline)] 5.1 .+-. 1.2 4.7 .+-. 2.4
3.7 .+-. 1.6 6.0 .+-. 2.2 5.1 .+-. 1.9 4.0 .+-. 1.7
cis-[PtCl.sub.2(NH.sub.3)(piperidine)] 7 2.6 .+-. 1.7 2.1 .+-. 1.2
1.3 .+-. 0.7 4.2 .+-. 1.7 3.1 .+-. 1.2 2.6 .+-. 0.7
trans-[PtCl.sub.2(NH.sub.3)].HCl 3 2 0.7 0.4 4.5 1.7 0.9
(4-piperidino-piperidine)
cis-[PtCl.sub.2(NH.sub.3)(piperazine)].HCl 15 >10 >10 5 NA NA
NA .sup.aIC.sub.50 in .mu.M, mean .+-. SD from at least 2
experiments.
Replacement of one NH.sub.3 group by either an aromatic-planar
amine (4-picoline) to give
trans-[PtCl.sub.2(NH.sub.3)(4-picoline)]or by an aliphatic
non-planar amine (piperidine) to give
trans-[PtCl.sub.2(NH.sub.3)(piperidine)] (1) enhanced the cytotoxic
activity relative to transplatin (Table 1). It should be noted that
complex (1) was more cytotoxic than the
[trans-[PtCl.sub.2(NH.sub.3)(4-picoline)] derivate which suggests
that activation of the trans position can be achieved by sterically
hindered amine ligands. Piperidine is more sterically hindered than
the 4-picoline because of the hydrogen atoms pointing out in
opposite directions in contrast to the planar hydrogens of the
aromatic ring of 4-picoline which may correlate with cytotoxic
activity.
In addition, replacing the second NH.sub.3 in
trans-[PtCl.sub.2(NH.sub.3)(4-picoline)] with piperidine to give
the mixed trans-[PtCl.sub.2(4-picoline)(piperidine)] (5) enhanced
the compound's cytotoxicity by a factor of 2 3 (Table 1). This
observation may be explained by a higher sterically hindered
structure of complex (5). The
trans-[PtCl.sub.2(4-picoline)(piperidine)] was 3 folds less active
than cisplatin and the is higher IC.sub.50 values of
trans-[PtCl.sub.2(4-picoline)(piperidine)] were consistent with the
lower level of Pt-DNA adducts as shown in FIGS. 1A and 1B.
The cytotoxicity of the sterically hindered compounds of the trans
geometry was compared with that of their cis counterparts
cis-[PtCl.sub.2(NH.sub.3)(4-picoline)] and
cis-[PtCl.sub.2(NH.sub.3)(piperidine)] (7). In contrast with the
effect of replacing one NH.sub.3 in the new trans-Pt complexes, a
similar replacement of one NH.sub.3 of cisplatin by an
aromatic-planar amine ligand (4-picoline) or by an aliphatic
non-planar amine (piperidine) resulted in lower cytotoxic activity
as compared to cisplatin itself. The complex
cis-[PtCl.sub.2(NH.sub.3)(piperidine)] is an analogue to the new
active cis-[Pt(NH.sub.3)(2-picoline)] (AMD473) a novel sterically
hindered anti-tumor compound designed to circumvent platinum drug
resistance and is currently under clinical trials.sup.(26).
The cytotoxicity of several of the piperazine-containing Pt
complexes on cisplatin sensitive and resistance cancer cell lines
was also determined. In particular, three pairs of cisplatin
sensitive and resistant cancer cell lines (A2780/A2780cisR,
41M/41McisR and CH1/CH1cisR) were employed.
The complexes were incubated for 24 hours with the above mentioned
cell lines and the cell survival in compound-treated cultures was
evaluated by the (microculture tetrazolium) MTS method as
previously reported. The results of the IC.sub.50 studies are shown
in Table 2A 2B.
TABLE-US-00002 TABLE 2A IC.sub.50 mean values (.mu.M) obtained for
several piperidine (pip) containing complexes (the number in
brackets represents resistance factor) A2780 A2780 cisR CH1 CH1cisR
41M 41McisR trans-PtCl.sub.2(4-pic)(pip) 9 .+-. 1 135 .+-. 7 (15)
26 .+-. 3 169 .+-. 9 35 .+-. 4 210 .+-. (6.0) (6.5)
trans-PtCl.sub.2(NH.sub.3)(4-pip) 5 .+-. 0.7 20 .+-. 2 (4.0) 15
.+-. 2 94 .+-. 6 27 .+-. 2 150 .+-. 8 (5.5) Cl.sub.2 (6.3)
trans-PtCl.sub.2(pip).sub.2 15 .+-. 2 250 .+-. 12 35 .+-. 2 280
.+-. 12 50 .+-. 3 300 .+-. 25 (6) (16.7) (8)
cis-PtCl.sub.2(pip).sub.2 15 .+-. 2 250 .+-. 12 32 .+-. 2 280 .+-.
12 50 .+-. 3 300 .+-. 25 (6) (16.7) (8)
cis-PtCl.sub.2(NH.sub.3)(pip) 26 .+-. 3 234 .+-. 17 (9) 36 .+-. 4
263 .+-. 17 64 .+-. 5 315 .+-. 24 (7.3) (4.9) Transplatin >200
>200 >200 >200 >200 >200 Cisplatin 2.2 38 6 23 26
107
TABLE-US-00003 TABLE 2B IC.sub.50 mean values (.mu.M) obtained for
several piperazine-(pz) containing complexes A2780 A2780cisR CH1
CH1cisR 41M 41McisR trans-[PtCl.sub.2(NH.sub.3)(pz)].HCl 5 .+-. 1
44 .+-. 4 (8.8) 12 .+-. 3 34 .+-. 4 (2.8) 52 .+-. 5 155 .+-. 12
(3.0) trans-[PtCl.sub.2(NBA)(pz)].HCl 16 .+-. 2 28 .+-. 2 (1.8) 17
.+-. 2 19 .+-. 3 (1.1) 32 .+-. 5 48 .+-. 3 (1.5)
trans-[PtCl.sub.2(IPA)(pz)].HCl 14 .+-. 1 30 .+-. 2 (2.1) 10 .+-. 1
50 .+-. 3 (5.0) 38 .+-. 3 122 .+-. 8 (3.2)
trans-[PtCl.sub.2(4-pic)(pz)].HCl 10 .+-. 3 24 .+-. 3 (2.4) 16 .+-.
2 42 .+-. 3 (2.6) 45 .+-. 3 147 .+-. 10 (3.3)
trans-[PtCl.sub.2(pip)(pz)].HCl 18 .+-. 2 64 .+-. 5 (3.6) 22 .+-. 3
85 .+-. 7 (3.9) 37 .+-. 4 118 .+-. 9 (3.2)
trans-[PtCl.sub.2(pz)(pz)].HCl 17 .+-. 3 43 .+-. 3 (2.5) 26 .+-. 2
53 .+-. 3 (2.0) 43 .+-. 3 153 .+-. 10 (3.6)
cis-[PtCl.sub.2(NH.sub.3)(pz)].HCl 10 .+-. 1 25 .+-. 2 (2.5) 28
.+-. 2 56 .+-. 3 (2.0) 46 .+-. 3 112 .+-. 12 (2.4)
trans-PtCl.sub.2(NH.sub.3).sub.2 >200 >200 >200 >200
>200 &- gt;200 cis-PtCl.sub.2(NH.sub.3).sub.2 2.2 .+-. 0.6
38 .+-. 3 (17.3) 6 .+-. 1 23 .+-. 3 (3.8) 26 .+-. 2 107 .+-. 8
(4.1) NBA = n-butylamine, IPA = isopropylamine, 4-pic =
4-methylpyridine, pip = piperidine, pip-piperazine. The numbers in
parentheses are the resistance factors (IC.sub.50
resistant/IC.sub.50 sensitive)
In terms of the SAR, replacing one or both amine ligands of
transplatin with piperazine markedly increases the antitumor
activity relative to transplatin indicating the positively charged
non-planar amine ligand (piperazine) can activate the trans
geometry. The most striking feature of these cytotoxicity studies
is that the complexes are at least as active as cisplatin against
the A2780cisR cell line that is resistant through enhanced DNA
repair/tolerance and elevated GSH levels. Especially noticeable are
the very low resistance factors (RF) of
trans-[PtCl.sub.2(NBA)(pz)].HCl (10) against all three cell lines
(RF<2) indicating efficient circumvention of cisplatin
resistance.
A possible explanation for the enhancement of anti-tumor activity
of these transplatin complexes is that the sterically hindered
ligands may decrease detoxification by thiols. The reduced
reactivity towards biological thiols and thioethers (proteins and
peptides) is considered beneficial since reaction of cisplatin with
biological sulfur containing ligands is believed to be in the
source of acquired resistance and the toxic side effects of the
drug.
Cellular Drug Uptake and DNA Platination
In order to determine drug accumulation in tumor cells, C-26 and
OV-1063 cells were exposed to the cytotoxic compounds
trans-[PtCl.sub.2(4-picoline)(piperidine)] (5) and
trans-[PtCl.sub.2(4-picoline)(piperazine).HCl] (13) for 24 hr and
compared with the drug uptake of cisplatin and transplatin under
the identical conditions. The Pt content associated with the cells
was measured by Atomic Absorption Spectroscopy (AAS). It was found
that trans-[PtCl.sub.2(4-picoline)(piperidine)] (5) penetrates the
cells very efficiently in both cell lines (6-fold higher than
cisplatin), as shown in FIG. 1A.
Also, compared to transplatin the penetration of
trans-[PtCl.sub.2(4-picoline)(piperidine)] (5) was 7-fold higher in
OV-1063 cells and 30-fold higher in C-26 cells, as shown in FIG.
1B). A time-dependent increase of
trans-[PtCl.sub.2(4-picoline)(piperidine)] (5) accumulation was
observed during the 4 (data not shown) to 24 hr of drug exposure.
The time-dependent accumulation of Pt in the cells was consistent
with the decrease in the IC.sub.50 values (Table 1). The
trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl (13) showed the
highest penetration values in both cell lines (22-fold higher in
compartment to cisplatin) (FIGS. 1A and 1B).
To determine the cellular DNA platination, C-26 cells and OV-1063
cells were exposed to these transplatin complexes for 4 hr or 24 hr
and compared with cisplatin and the platinum DNA content was
measured by AA spectrometer. The
trans-[PtCl.sub.2(4-picoline)(piperidine)] DNA platination was the
same as that of Cis-Pt in C-26 and OV-1063 cells and the values of
Pt molecules intercalated with DNA from
trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl complex were 7-fold
higher than of Cis-Pt in both cell lines (FIGS. 2A and 2B).
The formation of calf thymus DNA platination was also examined. To
this end, calf thymus DNA was incubated with different compounds
(Table 3) in which the following parameters were determined:
TABLE-US-00004 TABLE 3 DNA binding t.sub.1/2
.DELTA..DELTA..epsilon.max, .DELTA.T.sub.m, (0.C), r.sub.b = 0.05 %
IEC/ (min) r.sub.b = 0.05 0.01M Na.sub.+ 0.2 M unwinding adduct
cis-[PtCl.sub.2(NH.sub.3)(pip)] 113 0.93 -2.5 -6.0 13.2.degree. 5
Cis-[PtC.sub.2(NH.sub.3)(pz)] 35 3.5 -4.5 13.degree. 6
trans-[PtCl.sub.2(NH.sub.3)(pip)] 113 0.15 +7.6 -1.9 52.8.degree.
26 trans-[PtCl.sub.2(NH.sub.3)(pz)] 20 0.01 6.3 -1.9 26.4.degree.
13 trans-[PtCl.sub.2(4-pic)(pip)] 260 0.04 +5.5 +0.5 11.degree. 2
trans-[PtCl.sub.2(4-pic)(pz)] 12 -0.30 +4.7 -3.5 17.degree. 6
cis-[PtCl.sub.2(NH.sub.3)(pic)] 21 0.92 +1.6 -4.8 12.degree. 4
trans-[PtCl.sub.2(NH.sub.3)(4-pic)] 50 -0.29 +7.2 +0.6 39.6.degree.
40 trans-[PtCl.sub.2(NH.sub.3).sub.2] 100 -0.34 +8.7 +0.6
9.4.degree. 12 cis-[PtCl.sub.2(NH.sub.3).sub.2 100 1.27 -2.6 -4.0
13.degree. 6 t1/2 - half time (in minutes) of the binding of the
compounds to calf thymus DNA in 10 mM NaClO.sub.4, at 37.degree.
C., r.sub.i = 0.08 determined by differential pulse polarography;
.DELTA..sub..DELTA..epsilon.max - the maximum of the positive CD
band at around 275 nm, the difference between the control and
platinated calf thymus DNA; .DELTA.T.sub.m - the difference in the
melting temperature of unplatinated and platinated calf thymus DNA;
unwinding - the unwinding angle per adduct; % IEC/adduct -
frequency of interstrand crosslinks.
As can be seen, both piperazine- and piperidine containing
complexes bind to DNA at a significantly higher rate than
cisplatin.
Characterization of DNA Adducts by EtBr Fluorescence
EtBr, as the fluorescent probe, was used to distinguish between
perturbations induced in DNA by adducts of platinum (II)
compounds.sup.(14). Binding of EtBr by intercalation is blocked in
a stoichiometric manner by formation of the bifunctional adducts,
as of Cis-Pt, which results in a loss of fluorescence intensity. On
the other hand, formation of monofunctional adducts results only in
a slight decrease of EtBr fluorescence.
DNA platination measurements of DNA modified by
trans-[PtCl.sub.2(4-picoline) (piperazine)].HCl showed considerable
decrease in fluorescence which is in agreement with the formation
of bifunctional adducts. On the other hand, the decrease of
fluorescence intensity by adducts of
trans-[PtCl.sub.2(4-picoline)(piperidine)] was lower than that of
cisplatin, however, greater than that of transplatin. The best
adduct was formed with
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl (Table 4
and also in FIG. 3).
TABLE-US-00005 TABLE 4 Penetration and formation of Pt-DNA adducts
of various platinum complexes Penetration to cells Pt-DNA adducts
(pmol Pt/1 * 10.sup.6 (pmol Pt/50 .mu.g cells) 24 hr 24 hr DNA)
Name C-26 OV-1063 C-26 OV-1063 cis-DDP 2244 194 149 107 trans-DDP
384 474 0 0 trans-[PtCl.sub.2(4-pic)(pip)] 14974 2092 51 41
trans-[PtCl.sub.2(4-pic)(pz)].HCl 54461 12307 297 205
The conclusion thus drawn was that
trans-[PtCl.sub.2(4-picoline)(piperidine) forms monofunctional and
also bifunctional adducts that are capable of inhibiting the
intercalation of EtBr into the DNA and, therefore, decreasing of
EtBr fluorescence intensity.
In addition, DNA incubated with
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl (3) for 24
hr showed considerable decrease in EtBr fluorescence (slightly
higher than that of cisplatin) (FIG. 4). The difference between
complex (3) and cis-DDP was higher at low concentration, which
suggest that complex (3) posses higher affinity to DNA. The
decrease of fluorescence intensity by the adducts of
cis-[PtCl.sub.2(NH.sub.3)(piperaize)].HCl (8) was similar to that
of cisplatin (FIG. 4).
Assessment of Apoptosis
Apoptosis, also known as programmed cell death, is involved in the
regulation of cell number in multicellular organism, and the
pathogenesis of various diseases, including tumor progression,
neurodegenerative disorders and viral infections. It has been
demonstrated in most cell types that phosphatidylserine (PS), a
lipid normally confined to the inner leaflet of the plasma membrane
of the normal cell. In the cell that undergoes apoptosis the PS is
exported to the outer plasma membrane leaflet in the early stage of
apoptosis. PS exposure in treated C-26 and OV-1063 cells was
detected by staining with MC 540, which has a strong affinity to PS
and chromatin condensation was assessed by staining with DAPI, that
preferentially stains double stranded DNA.
Distinguishing features of apoptosis in
trans-[PtCl.sub.2(4-picoline)(piperidine)] treated OV 1063 cells
were observed as evidenced by appearance of red fluorescence in the
cell membrane and increasing green fluorescence of nucleus in
contrast to red-uncolored untreated cells (results not shown). The
results of this staining showed that large proportion of the
OV-1063 cells appeared to be apoptotic after 5 hr of treatment with
6.5 .mu.M of trans-[PtCl.sub.2(4-picoline)(piperidine)]. The cell
surface of C-26 cells became slightly red-fluorescent after 5 hr of
treatment with 4.5 .mu.M of
trans-[PtCl.sub.2(4-picoline)(piperidine)] (results not shown) in
contrast to none of the red fluorescence in untreated cells
(results not shown).
Recently, members of the caspase (CED-3/ICE) family of proteases
have been found to be crucial mediators of the complex biochemical
events associated with apoptosis.sup.(27). In particular, the
activation of caspase-3, which cleaves a number of different
proteins, including poly(ADP-ribose) polymerase (PARP), protein
kinase C.delta. and actin, has bean shown to be important for the
initiation of apoptosis.sup.(28). Thus, activation of caspase-3 was
measured in trans-[PtCl.sub.2(4-picoline)(piperidine)] treated C-26
and OV-1063 cells. It was found that OV-1063 cells treated for 5 hr
with 6.5 .mu.M trans-[PtCl.sub.2(4-picoline)(piperidine)] or with
7.5 .mu.M trans-[PtCl.sub.2[(4-picoline)(piperazine)].HCl activated
caspase-3 (.about.2-fold increase in fluorescence in treated cells
in comparison to untreated cells). Moreover, after 16 hr of
treatment of OV-1063 cells with
trans-[PtCl.sub.2(4-picoline)(piperidine)] or with
trans-[PtCl.sub.2[(4-picoline)(piperazine)].HCl there was a 3-fold
increase in fluorescence in the treated cells in comparison to
untreated cells (not shown).
To confirm that the observed fluorescent signal was due to
activation of caspase-3, the reversible Ac-DEVD-CHO inhibitor of
caspase-3-like proteases was added to the control and treated
samples. A drastic decrease in fluorescent signal was found in
samples treated with Ac-DEVD-CHO inhibitor (not shown), which
argues for specific activation of caspase-3. There was no
fluorescent signal found in C-26 cells treated with 4.5 .mu.M
trans-[PtCl.sub.2(4-picoline)(piperidine)].
To determinate whether OV-1063 or C-26 cells treated with cisplatin
undergo apoptosis these cell lines were treated for 5 or 16 hrs
with 2 .mu.M or 1.5 .mu.M, respectively. No fluorescent signal was
found in cisplatin treated OV-1063 cells or C-26 cells. These
findings are in agreement with data of L. Szmigiero et. al. which
demonstrated that there is no degraded DNA detected by agarose gel
electrophoresis in L1210 cells treated with cisplatin.sup.(29). It
was also in agreement with several findings which have shown that
colon cancer cells protect themselves by secreting a soluble
factor(s) that inhibit apoptosis.sup.(30) and by aberrant
activation of c-kit which protects colon carcinoma cells from
apoptosis.
Protein Binding
Since most platinum(II) derivatives which are administered
intravenously become protein bound within 24 hr, the binding
kinetics of two model proteins, Ubiquitin (MW 8565) and Heart
Myoglobin (MW 16951), to Pt complexes was determined. To this end,
a 1:1 reaction between the platinum complexes and the proteins were
carried out at 1 2 mM concentrations, in 10 mM phosphate buffer, pH
6.4 at 37.degree. C. Protein binding kinetics were measured
directly on the reaction mixtures, by Electrospray Ionization Mass
Spectrometry (ESI-MS). FIG. 5 shows that while the neutral
trans-PtCl.sub.2(NH.sub.3)(piperidine) binds rapidly to the
proteins, followed, with respect to binding rate, by cisplatin and
transplatin, the charged piperazine complexes had no significant
binding to the proteins. The combination of very rapid binding to
DNA with slow and inefficient binding to proteins is a very
desirable property of a platinum based anti-tumor drug.
Toxicity
In was found that
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl and cis-DDP
were non-toxic at concentration of 5 mg/kg, and
trans-[PtCl.sub.2(4-picoline)(piperazine)].HCl was non-toxic at
concentration of 20 mg/kg.
In Vivo Antitumor Effect
Female BALB/C mice (in the weight range of 17 20 gr) were injected
i.p. with 1*10.sup.6 C-26 colon carcinomas. The viability of these
cells was >90% by trypan blue exclusion.
The therapeutic efficacy of
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl was studied
and compared to cisplatin. Treatment was as performed according to
the schedule described above. The results are presented in Table 5
and in FIG. 6.
TABLE-US-00006 TABLE 5 Antitumor effect in first schedule of
treatment Dose Dose (mmole/ No. Survival Increase in Treatment
(mg/kg) kg) of mice (days) life span (%) control 7 11.5 .+-. 0.5
cisplatin 5 16.6 8 24 .+-. 5 108 trans-[PtCl.sub.2NH.sub.3 5 10.3 8
24 .+-. 3 108 (pip-pip)].HCl
EXAMPLE 3
Liposomal Formulation
Preparation and Characterization of Sterically Stabilized Liposomes
(SSL) Containing
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl
Preparation of SSL Containing trans-[PtCl.sub.2(NH.sub.3)
(piperidino-piperidine)].HCl
Trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl (10 mg/ml)
was dissolved in 0.9% NaCl at 65.degree. C. and left at this
temperature for 1 hr. Lipids (HSPC:cholesterol:PEG.sup.2000-DSPE
51:44:5) were dissolved in ethanol. The lipids were hydrated by
adding this ethanolic solution to the drug mixture. Final lipid
concentration was 150 mg/ml (15%) in 25% ethanol, at 65.degree. C.
The mixture was kept stirring for 1 hr at 65.degree. C., then
extruded at 65.degree. C., 5 times through 25 mm polycarbonate
filters with 200 nm pore size using Lipex extruder (Nothern Lipids
Inc, Vancouver, Canada), followed by extrusion 11 times trough a
100 nm pore size polycarbonate filter. Sized liposomes (.about.100
nm) were allowed to cool to room temperature. During the cooling, a
heavy precipitate formed the supernatant was collected. Then
supernatant was cooled to 4.degree. C. overnight and the
supernatant was collected again. The supernatant was collected and
dialyzed against 10 mM histidine buffer (pH=6.5) containing 10%
sucrose and 1 mM NaCl for overall of 5 times against 100 vols. of
buffer and 1 time against 200 vols at 4.degree. C. Under these
conditions, a complete equilibration with buffer should occur. The
final liposome dispersion was a translucent white.
Characterization of SSL Containing
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl
Liposomes were characterized for their size distribution at
25.degree. C. by dynamic light-scattering (DLS) with a Coulter
model N4 SD (Coulter Electronics, Hialeah, Fla., USA).
The concentration of the phospholipids (PLs) was checked by lipid
phosphorus content (modified Bartlett method).sup.(31).
The platinum concentration in the liposomes was measured by
flameless Zeeman atomic absorption spectrometer (FAAS). The
platinum concentration was calculated according to a calibration
curve included 5 standards of K.sub.2PtCl.sub.4 stock solution with
concentrations ranging from 50 to 250 ng platinum per mL.
The SSL containing
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl is
characterized by the following parameters: Size--102 nm;
Concentration of
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl in the
formulation: 1 mM; Concentration of the lipid in the formulation:
94 mM; and Percentage of encapsulation (Pt/Pl ratio in
liposomes/initial Pt/Pl ratio.times.100) 8%.
Characterization of Pt Release from
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl SSL
Sulfur containing glutathion (GSH) is known as strong platinophile.
Hence it was chosen for the release experiments of the platinum
from the liposome. Its fast reaction with platinum and the strong
chemical shift the binding of its sulfur induces on the
.sup.195Pt-NMR will enable us to detect only the
diaminedichloroplatinum at the range of interest. An active
positively charged derivate was
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl (3).
All NMR spectra were recorded on a Varian Inova 500 MHz
spectrometer using a 5 mm switchable probe. .sup.195Pt NMR spectra
were referenced externally to K.sub.2PtCl.sub.4 in HCl (-1624
ppm).
.sup.195NMR Experiment
To 0.5 mL of the liposome suspension (0.5 mg/mL) in NMR tube, 2 eq.
of glutathion (GSH) were added and the suspension was vigorously
shacked for 2 min. .sup.195Pt-NMR test indicated that the complex
inside the liposome is intact (.delta.=-2134.597 ppm). The sample
was left at 37.degree. C. .sup.195Pt-NMR follow up was done after
1, 2, and 7 days. Through the first 2 days the platinum moiety was
intact. .sup.195Pt-NMR that was done at the seventh day revealed
the total disappearance of the chemical shift characteristic of the
dimminedichloroplatinum(II) moiety.
To evaluate the effect of (GSH) on the release of the platinum drug
the above experiment was repeated with no GSH. .sup.195Pt-NMR
revealed that the complex is intact inside even after 10 day at
37.degree. C. (.delta.=-2132.585 ppm) with minor product at
.delta.=-2661.428 ppm.
In summery the results indicated clearly that the charged complex
trans-[PtCl.sub.2(NH.sub.3)(piperidino-piperidine)].HCl, in
contrast to cisplatin is released. The total disappearance of the
characteristic chemical shift of the diaminedichloroplatinum(II)
means that the ligands in the coordination sphere have changed.
Nevertheless, the fact that no change is apparent in the GSH free
experiment is not a clue for lack of release. For that the solution
out side should be filtered and atomic absorption (AA) and (if
possible) .sup.195Pt-NMR in order to verify the existence of the
platinum moiety outside the liposome.
EXAMPLE 4
Bis-platinum Tetra-functional Positively Charged Piperazine-based
Complexes
Continuing the efforts to synthesize non-classical platinum
complexes, tetrafunctional positively charged bis-platinum
complexes was synthesized according to the following scheme:
##STR00001##
Cis-PtCl.sub.2(BOC-Pz).sub.2 (1 g, 2.41 mmol) was dissolved in 20
mL DDW. To the stirred mixture, 2 eq. (0.9 g, 4,84 mmol) of
tert-butyl 1-piperazine carboxylate were added and the mixture was
wormed to 70.degree. C. Stirring and warming continued for 50 min.,
then yellow precipitate was collected and washed twice with 40 mL
DDW. After drying, the yellow product was characterized using
.sup.195Pt-NMR(DMF) and used with no further purification.
.sup.195Pt-NMR(DMF): .delta.=-2239.4 ppm, -2267.8 ppm
Synthesis of
Bis-[{trans,trans-(PtCl.sub.2-Pz).sub.2}(Linker)].2HCl
In dark, cis-PtCl.sub.2(Boc-Pz).sub.2 (538 mg, 1 mmol) was dissolve
in 50 mL DMF. To the stirred yellow solution, 1 eq. (169.88 mg, 1
mmol) of silver nitrate was added and the mixture was stirred at
room temperature for 48 hours. .sup.195Pt-NMR(DMF) indicated the
formation of the mono-nitrato/DMF mono-chloro diaminoplatinum(II)
(.delta.=-2002.987 ppm, -2123.995) with few traces of the starting
material at (.delta.=-2240.477 ppm) At his stage the AgCl
precipitate was filtered off and 0.5 eq. (110 mg, 0.5 mmol) of
4,7,10-trioxa-1,13-tridecanediamine were added. The mixture was
stirred at dark overnight. .sup.195Pt-NMR(DMF) shown the formation
of the mono-chloro-triamine platinum (.delta.=-2542.974 ppm,
-2570.911 ppm). The yellowish filtrate was taken and solvents were
evaporated under reduced pressure to dryness. The gum was dissolve
in 20 ml of ethanol and 1 mL of concentrated hydrochloric acid was
added. The mixture was stirred at room temperature until total
solubilization, and then temperature was elevated to reflux for 50
minutes. Through out this period of time yellowish precipitate was
formed. The reaction mixture was allowed to cool to room
temperature and the precipitate was collected, washed with 20 ml
ethanol and dried.
.sup.195Pt-NMR(H.sub.2O): .delta.=-2224.396 ppm, 2238.142 ppm,
2248.194 ppm.
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